Fractionating/collecting device of liquid chromatograph

ABSTRACT

In a preferred embodiment, a sample container storage part for storing a number of sample containers S, a nozzle for dropping a sample component separated and supplied by an LC and an additive liquid such as digestive fluid supplied from another liquid supplying part to the sample container S, a carrying mechanism for carrying and positioning the sample container at an arbitrary position under the nozzle, and a second nozzle, serving as a suction/injection mechanism, for sucking in the fractionated/collected sample component and injecting the sample component to another LC. The carrying mechanism provides a rotation mechanism. The carrying mechanism rotates over 180 degrees and carries the sample container S completed with fractionating/collecting to the position of the second nozzle, and the sample is sucked in by the second nozzle and injected to the LC of next stage.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to fractionating/collecting devicesfor fractionating and collecting a separated sample component in aliquid chromatograph, hereinafter also referred to as LC, performingseparation analysis of a solution sample.

[0003] 2. Description of the Related Art

[0004] An effluent from a liquid chromatograph is fractionated andcollected, and further analyzed in the liquid chromatograph or otheranalyzing devices such as a mass spectrograph (MS). There afractionating/collecting device for collecting onto a sample containersuch as a microplate is used.

[0005] One example of the mass spectrograph for analyzing the effluentfrom the liquid chromatograph is a Matrix Assisted LaserDesorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOFMS). Inthe MALDI-TOFMS, when fractionating and collecting sample componentsseparated in the liquid chromatograph on the MALDI-TOFMS analyzing plateusing the fractionating/collecting device, the sample components arecollected at a number of sites on a single plate. After the collectingprocess is completed, the plate is manually moved to the MALDI-TOFMSdevice for analysis.

[0006] As only one or two collecting plates can be used in theconventional fractionating/collecting devices, not many analyzingsamples can be prepared at night without any workers present, forexample, for such MALDI-TOFMS analysis.

[0007] Other analyzing methods for analyzing the effluent from theliquid chromatograph includes an LC for performing separation analysisthrough multiple stages, which include fractionating and collecting thesample component separated in the LC of the first stage with thetractionating/collecting device, and analyzing the collected samplecomponent in the LC of the second stage after processing. In theanalysis of protein, for example, in order to further analyze theprotein separated in the LC, a digestive enzyme is manually added to thedivided and collected sample component of the protein for decomposing topeptide, the peptide being further separated in another LC for analyzingin the MS, and analysis is carried out on the obtained MS data.

[0008] In the two-step separation task in the LC, manual tasks such asadding the digestive enzyme take a long time. Therefore, a liquidhandler capable of fractionating/collecting and dispensing is used forautomatically carrying out the task. Normally in the liquid handler, itsprobe moves to a fractionating/collecting position of the LC in thefirst stage to suck in the collected component, and then moves to aninjection port in the LC of the second stage to inject the suckedcomponent Thus, the space in the probe is large, which isdisadvantageous especially when a micro LC, for example, is used on thefractionating/collecting side to handle very small amounts of sample.

SUMMARY OF THE INVENTION

[0009] The first object of the present invention is to propose afractionating/collecting device capable of collecting samples on anumber of sample containers.

[0010] The second object of the present invention is to further proposea fractionating/collecting device capable of shortening time and savinglabor in handling the fractionated components between the first andsecond stages in a LC and which is capable of analyzing a small amountof fractionated component

[0011] The fractionating/collecting device according to the presentinvention for achieving the first object comprises afractionating/collecting mechanism for dropping and collecting aneffluent from a liquid chromatograph to a sample container from anozzle, a storage part for storing a number of sample containers, and acarrying mechanism comprising a vertical moving mechanism and ahorizontal moving mechanism for both carrying and positioning the samplecontainer to an arbitrary position under the nozzle.

[0012] In one aspect of the fractionating/collecting device of thepresent invention, multiple samples to be analyzed can be prepared sincesample collecting can be carried out on a number of sample containersand the sample containers can be stored by providing the storage partfor storing a number of sample containers and by configuring thecarrying mechanism so as to rotate within a range of the device and tomove freely in the vertical and horizontal directions. Furthermore, thecarrying mechanism is also used to position the sample container duringcollecting, and thus the device can be inexpensively realized.

[0013] Continuous analysis can also be carried out by combining theconventional fractionating/collecting device and a multi-purpose robotarm. However, in comparison with a device of the present invention, suchrobot arm does not only require to be a two-system robot arm whichincludes a carrying mechanism for carrying the plate and a collectionaiding mechanism for positioning the plate so that a sample liquid isdropped onto an appropriate position on the plate under the nozzledropping the effluent from the LC, but also requires a large area toinstall the robot arm.

[0014] If the nozzle is configured so that the effluent from the liquidchromatograph and a liquid from another liquid supplying part aresimultaneously dropped a matrix liquid, for example, and the effluentfrom the liquid chromatograph may be simultaneously dropped to form thedevice for preparing a sample for MALDI-TOFMS exclusively.

[0015] If the nozzle is configured so that either the effluent from theliquid chromatograph or a liquid from another liquid supplying part isselectively dropped only the effluent from the liquid chromatograph, forexample, may be dropped, and a desired liquid such as matrix liquid ordigestive fluid may be subsequently selectively dropped to form amultipurpose sample preparing device.

[0016] If the carrying mechanism is configured to be able to carry thesample container, in which sample collecting is completed, to apredetermined position out of the fractionating/collecting device, thesample container can not only be stored within the storage part but canalso be automatically loaded to an analyzing device.

[0017] If an outlet for discharging gas in the direction of the tipportion of the nozzle is provided at the periphery of the tip portion ofthe nozzle, it can prevent liquid from remaining at the tip portion ofthe nozzle.

[0018] When a sample fraction dropped onto the plate is dried andcrystallized, crystallization will not be even if the moisture contentwithin the collecting device under analyzing process changes. A port forexhausting gas provided in the vicinity of the nozzle can stabilizedrying of the fraction.

[0019] A fractionating/collecting device of the present invention forachieving the second object comprises a fractionating/collectingmechanism for dropping an effluent from liquid chromatograph to a samplecontainer with a nozzle, a carrying mechanism comprising a verticalmoving mechanism and a horizontal moving mechanism for both carrying andpositioning the sample container to an arbitrary position under thenozzle, a sample container storage part provided within a movable rangeof the sample container by the carrying mechanism for storing a numberof sample containers, and a suction/injection mechanism provided withina movable range of the sample container by the carrying mechanism forsucking in a sample in the sample container and injecting the suckedsample to another LC.

[0020] In another aspect of the present invention, as thefractionating/collecting device comprises a carrying mechanism forcarrying and positioning the sample container, and the mechanism forcarrying out suction and injection of samples for LC in the next stagewithin the movable range of the sample container, the LC provided withsuch fractionating/collecting device can carry out multi-stagechromatograph separation without any manual aid inexpensively andautomatically. Furthermore, automatization increases the rate ofoperation.

[0021] As the movable range of the suction/injection mechanism can berestricted by moving the sample container, dead space in the nozzlemechanism of the suction/injection mechanism can be decreased and verysmall amounts of fraction component can be handled.

[0022] The fractionating/collecting device of an open space type(non-closed configuration) has a risk of allowing a condensed water intothe sample container when, for example, the sample is being cooled downand thus it is not an ideal condition. If the fractionating/collectingmechanism, the carrying mechanism, the sample container storage part,and the suction/injection mechanism are stored within a sealed spaceprovided with a temperature control mechanism, it is advantageous forpreventing condensation when the sample is being cooled down.

[0023] If the nozzle for dropping the effluent from the LC is connectedto a liquid supplying part for dropping another liquid, for example, anadditive liquid such as digestive fluid simultaneously with the drop ofthe effluent, a device used exclusively for sample preparation tore-separate the fractionated/collected sample can be obtained.

[0024] If the nozzle for dropping the effluent from the LC is connectedto a liquid supplying part for arbitrary selecting and dropping anotherliquid at a different timing with the drop of the effluent, amultipurpose sample preparation device for adding an arbitrary additiveliquid to the fractionated/collected sample can be obtained.

[0025] Other aspects and advantages of the invention will becomeapparent from the following description, taken in conjunction with theaccompanying drawings, illustrating by way of example the principles ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention, together with aims and advantages thereof, maybest be understood by reference to the following description of thepresently preferred embodiments together with the accompanying drawingsin which:

[0027]FIG. 1 is a perspective view showing a configuration of oneembodiment of the present invention;

[0028]FIG. 2 is a cross sectional view showing a nozzle and mechanismsat the periphery of the nozzle of the embodiment in FIG. 1; and

[0029]FIG. 3 is a schematic diagram showing a configuration according toanother embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030]FIG. 1 is a schematic diagram showing a preferred embodiment ofthe present invention.

[0031] In FIG. 1, a fractionating/collecting device includes a carryingmechanism 3 arranged next to a mass spectrograph 12 for carrying andpositioning a sample container, and a storage part 6 arranged next tothe carrying mechanism 3 for storing a number of sample containers. Thefractionating/collecting device further includes a nozzle 22 arranged ata position not in the way of the carrying mechanism 3 for discharging amatrix liquid and an effluent from a liquid chromatograph, a port 20arranged in the vicinity of the nozzle 22 for supplying dry air, and atube 18 arranged at a periphery of a tip portion of the nozzle 22 forexhausting air in the direction of the tip portion. The tube 18 isconnected to a syringe (not shown).

[0032] The storage part 6 has shelves for storing a number of samplecontainers, and is able to store sample containers in which the samplecollecting is incomplete and the sample containers in which the samplecollecting is complete. The sample containers are, for example, for aMALDI-TOFMS sample plates.

[0033] The carrying mechanism 3 includes a holder 4 for holding thesample container, an X-R stage 2 which is provided with a drivingmechanism for moving the holder 4 within a plane (X direction), androtatable (R direction) and movable in the vertical direction (Zdirection), a mechanism (not shown) for supporting the X-R stage 2, arod screw 8 for moving the X-R stage 2 in the vertical direction, a Ystage 10, movable in the Y direction whithin a plane, for supporting therod screw 8 and a guide 16 for moving the Y stage 10 in the Y direction.

[0034] A supporter 30 of the X-R stage 2 has a screw that fits with therod screw 8. As the rod screw 8 rotates, the X-R stage 2 moves in thevertical direction.

[0035] A bearing 32 is provided to the supporter 30, allowing the X-Rstage 2 to be maintained in a predetermined direction independently fromthe rotation of the rod screw 8 and to rotate within the plane.

[0036] The Y stage 10 is provided with a mechanism for moving in the Ydirection along the guide 16, and a mechanism for rotating the rod screw8.

[0037] The sample container held to be carried by the carrying mechanism3 can be moved to and positioned at an arbitrary position within themovable range by the moving of the holder 4 in the X direction, thevertical moving as well as the rotation of the X-R stage 2, and themoving of the Y stage 10 in the Y direction.

[0038]FIG. 2 shows the nozzle 22 for discharging the liquid andmechanisms in the periphery thereof of the above embodiment.

[0039] A tube 26 for introducing the effluent from the liquidchromatograph and a tube 24 for introducing the matrix liquid areconnected to the nozzle 22. The tube 18 for introducing air is arrangedat the periphery of the nozzle 22. Since the nozzle 22 discharges verysmall amounts of liquid, the liquid sometimes remains at the tip portionof the nozzle 22 due to the surface tension, and can not besatisfactorily dropped onto the sample container. The air exhausted fromthe periphery of the tip portion of the nozzle 22 then stimulates theliquid to drop.

[0040] The port 20 arranged in the vicinity of the nozzle 22 forexhausting dry air blows the dry air to the sample liquid dropped ontothe sample container and dries the sample liquid.

[0041] The operation of the present embodiment will now be described.

[0042] First, the Y stage 10 of the carrying mechanism 3 is moved to apredetermined position of the storage part 6, the rod screw 8 is rotatedto move the X-R stage 2 in the Z direction for adjustment of height, theholder 4 is moved in the X direction from the X-R stage 2 to take outthe predetermined sample container.

[0043] After the sample container is taken out, the carrying mechanism 3moves the sample container to under the nozzle 22.

[0044] Under the nozzle 22, in order to collect the effluent from theliquid chromatograph and the matrix liquid dropped from the nozzle 22 ata predetermined position on the sample container, the carrying mechanism3 is fine-adjusted in the X and Y directions to be positioned at theliquid drop. After the positioning is finished, the liquid is dropped.The air exhausted from the tube 18 from the air discharging syringestimulates the liquid dropping.

[0045] When the liquid has been dropped, the dry air is blown from theport 20, and the dropped sample liquid is dried and crystallized.

[0046] The positioning, dropping and the drying processes are repeatedlycarried out until the fractionating/collecting of the relevant samplecontainer is complete.

[0047] After fractionating/collecting is completed, the carryingmechanism 3 carries the sample container to the storage part 6 again tostore the sample container in the storage part 6. Then the carryingmechanism 3 takes out a new sample container, carries the samplecontainer to under the nozzle 22, and repeats thefractionating/collecting process.

[0048] By repeating such processes, a number of sample containers inwhich fractionating/collecting is complete are stored in the storagepart 6.

[0049] When analyzing by mass spectrometry, the holder 4 takes out thesample container in which fractionating/collecting is complete from thestorage part 6, and the X-R stage 2 rotates over 180 degrees to move thesample container to a loading part 14 of the mass spectrograph 12 forloading.

[0050] The sample container in which the sample fractionating/collectingis complete may also be directly loaded from under the nozzle 22 to themass spectrograph 12.

[0051]FIG. 3 is a schematic diagram showing a configuration of anotherembodiment.

[0052] The fractionating/collecting device of this embodiment isprovided with a sample container storage part 6 capable of storing anumber of sample containers S such as a microplate, a nozzle 40 fordropping a sample component separated and supplied by the LC and anadditive liquid such as digestive fluid supplied from other liquidsupplying part to the sample container S, a carrying mechanism 3 forcarrying and positioning the sample container S at an arbitrary positionunder the nozzle 40, and a nozzle 60, which serves as asuction/injection mechanism, for sucking in the fractionated/collectedsample component and injecting the sample component to another LC in ahousing F shown with a dashed line. The housing F has a temperaturecontrol function to maintain its internal space at a predeterminedtemperature, and has a sealed configuration in which the internal spaceis externally blocked.

[0053] The sample container storage part 6 has shelves for storing anumber of sample containers, and both the sample container in which thesample collecting is incomplete and the sample container in which thesample collecting is complete can be stored.

[0054] The carrying mechanism 3 is the same as that shown in FIG. 1 andthus the description thereof shall be omitted.

[0055] The nozzle 40 is arranged in the vicinity of the carryingmechanism 3 so as not to be in the way of the sample container storagepart 6. A tube for sending the effluent from the LC, and a tube forsending an additive liquid 52 such as digestive enzyme or a reactiveliquid are connected to the nozzle 40.

[0056] In the LC, an eluate 50 is supplied by a pump 48 and sends asample which is injected from an injector 46, to a column 44. The sampleseparated in the column 44 is detected in a detector 42 and is droppedfrom the nozzle 40 and fractionated/collected at the sample container S.

[0057] Furthermore, the additive liquid 52 is supplied by a pump 49, andis dropped from the nozzle 40 to the sample container S. The additiveliquid 52 can be dropped onto the sample container S simultaneously withthe effluent from the LC by having the pump 49 continuously supplied theliquid, or can be dropped after the effluent from the LC is dropped byhaving the pump 49 supplied the liquid over a predetermined time,.

[0058] The nozzle 60 is arranged within a movable range of the samplecontainer S at a position different from the nozzle 40. In thisembodiment, the nozzle 60 is arranged on the side opposite the nozzle 40with respect to the carrying mechanism 3. The nozzle 60 is movablysupported so as to be able to suck in the sample in the sample containerS and inject the sucked sample into an injection port 54 placed withinthe housing F.

[0059] The injection port 54 is connected to an injector 46 a of the LCof the next stage, and injects the sample to the LC of the next stage byway of the injector 46 a. In the LC of the next stage, the eluate 52 ais supplied by a pump 48 a, and sends the sample, which is injected fromthe injector 46 a, to a column 44 a. The sample component separated inthe column 44 a is supplied to the MS, which is acting as a detector, tobe detected.

[0060] The operation of the present embodiment shall now be described.

[0061] First, the carrying mechanism 3 moves to the position of thesample container storage part 6, and the holder 4 moves in the Xdirection to take out the sample container S. The sample container Staken out is carried to under the nozzle 40, and the effluent from theLC and the additive liquid. 52 such as digestive enzyme are droppedthereon from the nozzle 40. The collecting task is carried out bypositioning the sample container in the X and Y directions by means ofthe carrying mechanism 3 so that the effluent 50 and the additive liquid52 are dropped to an appropriate location on the sample container S.

[0062] The sample container S, in which the sample collecting iscomplete, is stored in the sample container storage part 6, a new samplecontainer S is taken out from the sample container storage part 6, andthe collecting of the new sample is repeated. In the sample containerstorage part 6, the collected samples are maintained at a constanttemperature.

[0063] The sample container S completed with sample collecting andstored in the sample container storage part 6 is taken out as neededfrom the sample container storage part 6 by means of the carryingmechanism 3. Then the X-R stage 2 is rotated over 180 degrees with therod screw 8 as the center and is further adjusted in height to positionthe sample container S under the nozzle 60. The nozzle 60 sucks in thesample in the sample container S, moves to the injection port 54 placednearby and injects the sucked sample to the injection port 54.

[0064] The sample injected to the injection port 54 is sent to thecolumn 44 a with the eluate 52 a supplied by the pump 48 a from theinjector 46 a, and is further separated and sent to the MS fordetection.

What is claimed is:
 1. A fractionating/collecting device comprising: afractioning/collecting mechanism for dropping and collecting an effluentfrom liquid chromatograph onto a sample container with a nozzle; astorage part for storing a number of sample containers; and a carryingmechanism provided with a vertical moving mechanism and a horizontalmoving mechanism to carry and position the sample container at anarbitrary position under the nozzle.
 2. The fractioning/collectingdevice according to claim 1, wherein the horizontal moving mechanismprovides a mechanism for rotating a part holding the sample containerwhithin a plane, and is configured to move the sample container to adetermined position out of the fractioning/collecting device.
 3. Thefractioning/collecting device according to claim 1, wherein the nozzleis configured so as to simultaneously drop the effluent from the liquidchromatograph and a liquid from another liquid supplying part.
 4. Thefractioning/collecting device according to claim 1, wherein the nozzleis configured so as to selectively drop one of either the effluent fromthe liquid chromatograph or a liquid from another liquid supplying part.5. The fractioning/collecting device according to claim 1, wherein anoutlet for blowing gas in a direction of the tip portion of the nozzle,is provided at a periphery of a tip portion of the nozzle.
 6. Thefractioning/collecting device according to claim 1, wherein a port forexhausting gas is arranged in the vicinity of the nozzle, the exhaustedgas drying the collected sample.
 7. A fractioning/collecting devicecomprising: a fractioning/collecting mechanism for dropping andcollecting an effluent from liquid chromatograph onto a sample containerwith a nozzle; a carrying mechanism providing a vertical movingmechanism and a horizontal moving mechanism for carrying -and positionthe sample container at an arbitrary position under the nozzle; a samplecontainer storage part arranged within a movable range of the samplecontainer by the carrying mechanism for storing a number of samplecontainers; and a suction/injection mechanism arranged within a movablerange of the sample container by the carrying mechanism for sucking in asample in the sample container and injecting the sucked sample intoanother liquid chromatograph.
 8. The fractioning/collecting deviceaccording to claim 7, wherein the fractioning/collecting mechanism, thecarrying mechanism, the sample container storage part and thesuction/injection mechanism are arranged in a sealed space provided witha temperature control mechanism.
 9. The fractioning/collecting deviceaccording to claim 7, wherein the nozzle is connected to a liquidsupplying part for dropping a different liquid simultaneously with thedropping of the effluent from the liquid chromatograph.
 10. Thefractioning/collecting device according to claim 7, wherein the nozzleis connected to a liquid supplying part for dropping a different liquidarbitrarily selected at a different timing with the effluent from theliquid chromatography.